P4–150: Preliminary characterization and structural analysis of the prion protein Homolog, Shadoo

Background: Prion diseases including Mad Cow Disease (BSE) and Creutzfeldt-Jakob Disease (CJD) are disorders of protein folding. In prion infections, the normal cellular prion protein (PrPC) is refolded into an infectivity-associated form (PrPSc). Within the larger picture of prion genesis, another CNS-expressed gene may also need to be considered: Sprn, encoding the hypothetical protein Shadoo (Sho). Sequence analysis revealed the protein to comprise an N-terminal signal sequence, an Arg-rich basic region encompassing up to six tetrarepeats; a hydrophobic central domain of 20 residues with strong homology to PrP; a less conserved C-terminal domain containing one N-linked glycosylation site; and a putative glycophosphatidylinositol (GPI)-anchor attachment site. Objective(s)/Methods: We have cloned, expressed and purified His-tagged mouse Sho in E. coli, from a plasmid encompassing the basic repeats, the hydrophobic patch and the Cterminus, pHis10MoSho(25-122). Results: SDS-PAGE and western blotting revealed that recombinant His10MoSho(25-122) protein was over-expressed as a 14-kDa band. Approximately 70% of Sho polypeptides were present in a supernatant fraction, indicating that the recombinant protein was soluble in the cytosol. His10Sho(25-122) was purified by Ni2 affinity chromatography to 80% purity, and close to 90% purity with a further size-exclusion chromatographic step. However, a band of a molecular weight size of 12-kDa was visible during both purification procedures. An interstitial deletion of 20 residues designed to obviate this putative endoproteolytic event resulted in the production of a single polypeptide species of 13-kDa. Circular Dichroism (CD) detected a high population of random-coiled structure in MoSho(25-122) while the internally deleted form displayed prominent beta-sheet content. Conclusions: These results suggest internal residues in mouse Sho may modulate beta-content and have an important functional role. We conclude that Sho may comprise a new point of entry for deciphering conformational transitions within cellular prion proteins.